b-Amyloid protein converting enzyme 1 and brain-specific type II membrane protein BRI 3 : binding partners processed by furin Louise Wickham,* Suzanne Benjannet,* Edwige Marcinkiewicz,* Michel Chretien  and Nabil G. Seidah* *Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Quebec, Canada  Regional Protein Chemistry Center, Diseases of Ageing Unit, Ottawa Health Research Institute, Ottawa, Ontario, Canada Abstract Using a yeast two-hybrid system, we screened a human brain cDNA library for possible interacting proteins with the C-ter- minal cytosolic tail of the b-secretase b-amyloid protein con- verting enzyme (BACE)1. This identified seven potential candidates, including the brain-specific type II membrane protein BRI 3 . Co-localization and co-immunoprecipitation experiments confirmed that BACE1 and BRI 3 co-localize and interact with each other via the cytosolic tail of BACE1. Furthermore, pulse and pulse-chase analyses revealed that the pro-protein convertases furin, and to a lesser extent PC7 and PC5A, process BRI 3 into a C-terminal secreted  4-kDa product. Thus, furin efficiently processes both pro-BACE1 and its novel interacting protein pro-BRI 3 . Keywords: amyloid precursor protein, Beta secretase, binding partners, BRI 3 , cytosolic tail, two-hybrid system. J. Neurochem. (2005) 92, 93–102. In Alzheimer’s disease (AD), the amyloidogenic pathway generating the amyloid peptide Ab starts by b-secretase cleavage of b-amyloid precursor protein (bAPP) at the EVKM 652 flDA sequence. This results in the generation of a membrane-bound  10-kDa 99-amino acid peptide, known as C99. Five different groups simultaneously reported the isolation and initial characterization of the human aspartyl proteinase b-secretase (reviewed in Dominguez et al. 2001; Vassar 2001). Two candidate b-amyloid protein converting enzymes were initially proposed, BACE1 and BACE2. Both are type I membrane-bound aspartyl proteinases with a pro- domain. In the case of BACE1 this pro-domain is rapidly cleaved intracellularly for maximal activation of the prote- inase. We (Benjannet et al. 2001) and others (Bennett et al. 2000; Creemers et al. 2001) have demonstrated that the convertases furin and pro-protein convertase (PC)5 are the major enzymes involved in the processing of pro-BACE1 into active BACE1, through cleavage at the RLPR 45 fl sequence. The presence of the pro-segment of BACE1 was shown to be important for both the cellular traffic of BACE1 toward the Golgi (Benjannet et al. 2001) and regulation of its activity (Shi et al. 2001). The zymogen processing of pro- BACE2 was shown to be autocatalytic (Hussain et al. 2001). Both BACE1 and BACE2 cleave at the b and other sites in bAPP. For example, BACE1 can also cleave bAPP at an internal Y10flE 11 b¢ site within Ab (Vassar et al. 1999). Within the endoplasmic reticulum (ER), b-site proteolysis predominates, whereas in the trans-Golgi network (TGN), b¢ cleavage is favored. (Huse et al. 2002). BACE2 preferen- tially cleaves Ab at the VF 19 flF 20 flAE site, probably acting as an alternative a-secretase (Yan et al. 2001). We recently showed that although BACE1 expression is abundant in the brain cortex and hippocampus, co-localizing with bAPP, BACE2 is poorly expressed in the brain except in the olfactory lobe (mitral cells), cerebellum (Purkinje cells) and cortical layers (Marcinkiewicz and Seidah 2000). Received June 30, 2004; revised manuscript received August 25, 2004; accepted August 26, 2004. Address correspondence and reprint requests to Nabil G. Seidah, Clinical Research Institute of Montreal, 110 Pine Avenue West, Mon- treal, Quebec, H2W 1R7, Canada. E-mail: seidahn@ircm.qc.ca Abbreviations used:Ab, amyloid b-peptide; AD, Alzheimer’s disease; APLP, amyloid precursor-like protein; BACE, b-amyloid protein con- verting enzyme; bAPP, b-amyloid precursor protein; BRI 3 , brain-specific type II membrane protein; CT-BACE1, cytosolic tail of BACE1; CTF- BRI 3 , C-terminal fragment of BRI 3 ; EGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; mAb, monoclonal antibody; NARC, neural apoptosis regulated convertase; PACE, paired basic amino acid converting enzyme; PAGE, polyacrylamide gel electro- phoresis; PC, pro-protein convertase; PLSCR1, phospholipid scramblase 1; SDS, sodium dodecyl sulfate; SKI, subtilisin-kexin isozyme; TGN, trans Golgi network. Journal of Neurochemistry , 2005, 92, 93–102 doi:10.1111/j.1471-4159.2004.02840.x Ó 2004 International Society for Neurochemistry, J. Neurochem. (2005) 92, 93–102 93